Multiepitope fusion antigens and vaccines and their use in treatment of enterotoxigenic diarrhea

ABSTRACT

Provided herein are polypeptides comprising up to 9 antigenic elements of ETEC virulence determinants: 7 CFA adhesins [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, and 2 toxins expressed by all ETEC strains, were genetically fused together for CFA-toxoid fusion with proteins (CFA/I/II/IV-STa-toxoid-LTtoxoid). Methods for making these polypeptides and their use in the treatment of ETEC related disease are also provided.

REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/105,195, filed Jun. 16, 2016, now U.S. Pat. No. 10,646,560, issuedMay 12, 2020, which is a 35 U.S.C. § 371 U.S. national entry ofInternational Application PCT/US2014/070874, having an internationalfiling date of Dec. 17, 2014, which claims the benefit of U.S.Provisional Patent Application No. 61/917,105, filed on Dec. 17, 2013,the content of each of the aforementioned applications is hereinincorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 12, 2014, isnamed P12168-02_ST25.txt and is 30,350 bytes in size.

BACKGROUND OF THE INVENTION

Virulence heterogeneity among bacterial or viral strains or isolatesremains as one major challenge in vaccine development. Like otherinfectious pathogens, enterotoxigenic Escherichia coli (ETEC) strainsare genetically and immunologically heterogeneous. ETEC strains are themost common bacteria causing diarrhea, a disease continues to be thesecond leading cause of death to children younger than 5 years indeveloping countries and remains as a major threat to global health.These ETEC strains produce immunologically different colonization factorantigens (CFAs) to colonize host small intestines and enterotoxins todisrupt fluid and electrolyte homeostasis in small intestinal epithelialcells that leads to fluid hyper-secretion and diarrhea. There are 23 CFAor CS (coli surface antigen) adhesins and 2 very distinctiveenterotoxins (heat-labile toxin, LT, and heat-stable toxin type Ib, STa)characterized from ETEC strains associated with human diarrhea. As ETECstrains expressing any one or two of these adhesins with eitherenterotoxin can cause diarrhea, developing vaccines to effectivelyprotect against ETEC diarrhea continues to be challenging.

It has been observed that immunity induced by individual antigens lackedin cross protection against ETEC, clearly due to immunologicalheterogeneity among CFAs and toxins. Early experimental vaccine studiesshowed that candidates carrying a single adhesin and/or toxin antigeninduced immunity protecting against only ETEC strains expressing thehomologous adhesin or toxin. The first ETEC vaccine candidate, killedETEC prototype strain H10407 (078:H11, LT+/STa+/CFAI+), inducedanti-CFA/I and anti-LT immunity and protected against homologouschallenge. Similarly to homologous protection from anti-CFA immunity,anti-LT antitoxin immunity protected against ETEC strains expressing theLT toxin but not against ETEC strains expressing STa toxin. Oralwhole-cell ETEC vaccine candidates currently under development includerCTB-CF and ACE527. The rCTB-CF is a killed cocktail product of 5strains expressing 6 CFA adhesins and recombinant B subunit of choleratoxin (CT) which is a homologue of LT. ACE527 carried 3 live attenuatedE. coli strains that express 5 CFA adhesins, 1 CFA adhesin subunit, andLTB subunit. The killed rCTB-CF induced antibody responses protectingagainst 70% ETEC infection or against moderate to severe diarrhea toadults from developed countries travelling to ETEC endemic countries.This product, however, caused adverse effects and provided nosignificant protection against ETEC diarrhea when given to childrenespecially very young children living in endemic areas, or failed toreduce overall diarrhea incidences among adult travelers. The liveattenuated ACE527 initially showed some adverse effects, but theseadverse effects were reduced or eliminated when a lower dose was used.ACE527 was shown to induce antibody responses to LTB, CFA/I, CS3, andCS6 among adult volunteers and to protect against severity of diarrheaoutcome from homologous challenge.

Although efforts are taken to improve both products, these two candidateproducts were not optimal in providing satisfactory protection againstETEC diarrhea. Both cocktail products carry no STa antigens to induceantibody response against STa toxin, and require a relatively high dosefor oral administration in order to induce host immune responses againsteach target adhesin and LT toxin. A high administration dose tends tocarry excessive somatic antigens, particularly lipopolysaccharide (LPS),which likely cause vomiting among very young vaccine receipients and maymask stimulation of host immune responses specifically to adhesins andtoxin. The inability to induce anti-STa antibody response apparently isanother cause of lacking in effective protection, as anti-LT_(B) (oranti-CT_(B)) immunity induced by either product may protect only againststrains expressing LT toxin but not against STa⁺ ETEC strains. STa⁺ ETECstrains are associated with over two thirds of human ETEC diarrhea casesand moderate to severe ETEC diarrhea cases, and are also a leading causeof diarrhea to children younger than 3 years who live in developingcountries. Therefore, ETEC vaccines need also to induce protectiveanti-STa immunity in order to provide effective protection against ETECdiarrhea.

However, the 19 amino-acid STa is poorly immunogenic and potently toxic;thus itself cannot induce anti-STa immunity, nor could it be a safeantigen even if it were immunogenic.

Therefore, there still exists an unmet need to produce a vaccine whichcan elicit an immune response and can also induce protective immunity tomultiple CFA antigens, as well as anti-LT and anti-STa immunity thatwould be broadly protective against ETEC.

SUMMARY OF THE INVENTION

In accordance with an embodiment, the present invention provides a multiepitope fusion antigen (MEFA) comprising a polypeptide molecule encodingthe colonization factor antigens (CFA) antigens CFA/I, CFA/II (CS, CS2,CS3), CFA/IV (CS4, CS5, CS6).

In accordance with another embodiment, the present invention provides aMEFA comprising a polypeptide molecule encoding the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6), and further comprising one or more enterotoxins, or fragmentsthereof, covalently linked to the polypeptide molecule.

In accordance with a further embodiment, the present invention providesa MEFA comprising a polypeptide molecule encoding the colonizationfactor antigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV(CS4, CS5, CS6), and further comprising heat-labile toxin (LT) andheat-stable toxin (STa).

In accordance with an embodiment, the present invention provides anucleic acid molecule encoding the polypeptide molecules describedherein.

In accordance with another embodiment, the present invention provides anexpression vector comprising the nucleic acid molecule encoding thepolypeptide molecules described herein.

In accordance with a further embodiment, the present invention providesmicro-organism transformed with the expression vector described herein.

In accordance with an embodiment, the present invention provides avaccine composition comprising the polypeptide molecules describedherein, and a pharmaceutically acceptable carrier.

In accordance with another embodiment, the present invention provides amethod for the therapy or prophylaxis of Enterotoxigenic Escherichiacoli (ETEC), infection in a subject comprising administering to thesubject the polypeptide molecules or the vaccines described herein.

In accordance with a further embodiment, the present invention providesa method for the therapy or prophylaxis of diarrhea in a subjectcomprising administering to the subject the polypeptide molecules or thevaccines described herein.

In accordance with still another embodiment, the present inventionprovides a method for the therapy or prophylaxis of EnterotoxigenicEscherichia coli (ETEC), infection in a subject comprising administeringto the subject the polypeptide molecules or the vaccines describedherein and administration of at least one additional therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-ID. Construction and detection of an embodiment of theinvention comprising ‘CFAI/II/IV-STa-_(toxoid)-LT_(toxoid)’ MEFA. (A)Construction of the CFAI/II/IV MEFA. The most antigenic epitopes of theCS1, CS2, CS3, CS4, CS5 and CS6 major structural subunits were embeddedinto CFA/I major subunit by replacing the CfaB surface-exposed but lessantigenic epitopes. (B) Construction of the 3xSTa_(N12S)-LT_(toxoid)fusion. Three copies of the STa_(toxoid) STa_(N12S) gene weregenetically fused to the monomeric LT_(toxoid) (LT_(R192G/L211A)) geneusing SOE (splicing overlap extension) PCRs. (C) Construction ofCFA-toxoid MEFA. A substitution of the first 150 amino acids of the3xSTa_(N12S)-LT_(toxoid) (the N-terminal STa_(N12S) and the first 131amino acids of LTA subunit) with the CFA/I/II/IV MEFA created theCFA/I/II/IV-STa_(N12S)-dmLT MEFA. Four linkers: LGA, GPVD (SEQ ID NO:36), Gly-Pro linker GPGP (SEQ ID NO: 37), and L-linker (DPRVLSS, SEQ IDNO: 39) were used for the construction. (D) Western blot to detect theCFA/I/II/IV-STa_(N12S)-LT_(toxoid) MEFA protein with anti-CFA/I,anti-CS1, -CS2, -CS3, -CS4, -CS5, and anti-CS6 MAb hybridoma supernatant(1:100; provided by Dr. AM Svennerholm), and rabbit anti-CT (1:3300;Sigma) and anti-STa antiserum (1:3300; provided by Dr. DC Robertson).Extracted MEFA proteins separated in 12% PAGE gel were detected witheach anti-adhesin MAb, anti-CT and anti-STa antiserum and IRDye-labeledgoat anti-mouse IgG or anti-rabbit IgG (1:5000; LI-COR). Lane (+)indicated the CFA/I/II/IV-STa_(N12S)-LT_(toxoid) MEFA proteins, whereaslane (−) of extracted total proteins of E. coli BL21 host strain as thenegative control. Lane M is the protein marker (in kilodaltons;Precision Plus Protein pre-stained standards; Bio-Rad).

FIG. 2. Mouse serum anti-adhesin and antitoxin IgG antibody titers.Anti-CFA/I, anti-CS1, -CS2, -CS3, -CS4/CS6 and anti-CS5/CS6, andanti-STa and anti-LT IgG antibodies in the serum of each mouse immunizedwith ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’ fusion antigen (solid circles)and the serum of each control mouse (blank circles) were titrated inELISAs. CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 adhesin heat-extractedfrom E. coli or ETEC strains in Table 1 (500 ng per well of a 2HBplate), STa-ovalbumin (10 ng per well of a Costar plate), or LT (ListBiological Laboratories, Inc.; 100 ng per well of a 2HB plate) andHRP-conjugated goat-anti-mouse IgG (1:3300; the secondary antibodies)were used to titrate IgG antibodies specific to CFA/I, CS1, CS2, CS3,CS4/6, CS5/6 adhesins and to STa and LT toxins, respectively. Theantibody titer was calculated from the highest dilution of a serumsample that produced an ELISA optical density of greater than 0.3 (abovethe background) and presented in a log₁₀ scale. Each dot represented amouse IgG titer, and the bars indicated the mean titer of the group.

FIG. 3 depicts the titration of anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS6,-CS5/CS6, and anti-CS6 IgA antibodies in fecal suspension samples of theimmunized mice (solid dots) and control mice (circles). Five hundrednanogram CFA adhesins heat-extracted from E. coli strains expressingCFA/I, CS1, CS2, CS3, CS4/CS6 or CS5/CS6 were coated to each well of anImmulon 2HB plate. Fecal suspension sample from each mouse (1:20dilution), in triplicate, was added to each well. HRP-conjugatedgoat-anti-mouse IgA (1:1000) was used as the secondary antibodies.Optical densities were used to calculate anti-STa antibody titers (inlog₁₀).

FIG. 4 shows CFA multiepitope fusion antigen (MEFA) nucleic acid andamino acid sequences of an embodiment of the present invention.

FIG. 5 depicts the construction and detection of the‘CFAI/II/IV-STa_(toxoid)-LT_(toxoid)’ fusion. Panel A: construction of‘CFAI/II/IV-STa_(toxoid)-LT_(toxoid)’ (CFA-toxoid) multiepitope fusionantigen from a CFA MEFA fusion antigen and toxoid fusion3xSTa-LT_(toxoid). Replacement of the first 170 amino acids of the3xSTa_(A14Q)-tmLT (Zhang et al., Clin Vaccine Immunol., 2014 February;21(2):243-9) or 3xSTa_(N12S)-LT_(toxoid) with a CFA multiepitope fusionantigen yielded CFA-toxoid fusions ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’and ‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’. Panel B: Detection of“CFA/I/II/IV-STa_(A14Q)-dmLT’ in Western blot with anti-CFA/I MAbhybriboma supernatant (1:100; provided by Dr. AM Svennerholm), anti-CTserum (1:3300; Sigma), and anti-STa antiserum (1:3300; provided by Dr.DC Robertson). IRDye-labeled goat anti-mouse IgG or anti-rabbit IgG(1:5000; LI-COR, Lincoln, Nebr.) was used as the secondary antibodies.

FIG. 6 shows the titration of anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS6,-CS5/CS6, —STa, and anti-LT IgG antibodies in serum samples of the miceimmunized with ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’ fusion antigen(solid circles) and the control mice (blank circles). Five hundrednanograms of CFA adhesins heat-extracted from E. coli strains expressingCFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6, 10 ng STa-ovalbumin, or 100 ngLT (List Biological Laboratories, Inc.) were coated to each well of anImmulon 2HB plate or a Costar plate to titrate anti-CFA/I, -CS1, -CS2,-CS3, -CS4/CS6, -CS5/CS6, —STa, and anti-LT IgG antibodies,respectively. Serum samples from each mouse (1:200 dilution) were addedto each well (in triplicate). HRP-conjugated goat-anti-mouse IgG(1:3000) was used as the secondary antibodies. Optical densities ofgreater than 0.3 (after subtracting the background reading) were used tocalculate anti-STa antibody titers (in log₁₀).

FIG. 7 shows the titration of anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS6,-CS5/CS6, —STa, and anti-LT IgG antibodies in serum samples of the miceimmunized with CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’ fusion antigen (solidcircles) and serum samples collected prior to immunization (blankcircles).

FIG. 8 depicts the titration of anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS6,-CS5/CS6, —STa, and anti-LT IgG antibodies in serum samples of the miceco-administrated with the e CFA MEFA and ‘3xSTa_(N12S)-LT_(toxoid)’fusion (solid circles) and serum samples collected prior to immunization(blank circles).

FIG. 9 depicts mouse serum in vitro antibody neutralization activityagainst STa toxin. Intracellular cyclic GMP concentration (pmole/ml) inT-84 cells incubated with STa toxin and mouse serum was measured with anEIA cGMP ELISA kit (Assay Design) and was used to indicate anti-STaantibody neutralizing activity. As STa toxin elevates intracellular cGMPin T-84 cells, while neutralizing anti-STa antibodies neutralize thetoxin and prevent STa from stimulating cGMP, a lower cGMP concentrationindicates a stronger neutralization activity of anti-STa antibodies. Theserum sample (30 μl; in a final dilution of 1:33.3) pooled from eachgroup of mice immunized with ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’,‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’, co-immunized with ‘CFA/I/II/IV‘and’3xSTa_(N12S)-LT_(toxoid)’, the control group, or the serum samplecollected prior to immunization was incubated with STa toxin (2 ng, in150 μl cell culture medium) for 30 minutes at room temperature, and theserum-toxin mixture was added to T-84 cells (1 ml of final volume withcell culture medium). Intracellular cGMP concentration in T-84 cells wasmeasured after 1 hour incubation at a CO₂ incubator, with the mean cGMPand standard deviation (from four to six replicates) of each groupindicated as columns and bars. The cGMP in T-84 cells cultured with cellculture medium alone (without STa toxin or serum; no STa toxicity), orwith STa toxin in culture medium (without serum; STa toxicity) were usedas references.

FIG. 10 shows the nucleic acid sequence of a CFA-toxoid multiepitopefusion antigen (CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)) gene of anembodiment of the present invention.

FIG. 11 shows the translated amino acid sequence of a CFA-toxoidmultiepitope fusion antigen protein(CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)).

FIG. 12 depicts the amino acid sequence of a CFA-toxoid multiepitopefusion antigen protein (CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)) having 435amino acids with a predicted molecular weight of 47.56 kilodaltons.

FIG. 13 shows the nucleic acid sequence of a CFA-toxoid multiepitopefusion antigen (CFA/I/II/IV-STa_(N12S)-LT_(toxoid)) gene of anembodiment of the present invention.

FIG. 14 shows the translated amino acid sequence of a CFA-toxoidmultiepitope fusion antigen protein(CFA/I/II/IV-STa_(N12S)-LT_(toxoid)).

FIG. 15 depicts the amino acid sequence of a CFA-toxoid multiepitopefusion antigen protein (CFA/I/II/IV-STa_(N12S)-LT_(toxoid)) having 435amino acids with a predicted molecular weight of 47.56 kilodaltons.

FIG. 16 shows mouse serum in vitro antibody neutralization activityagainst cholera toxin (CT). Intracellular cAMP concentrations (pmole/ml)in T-84 cells measured with an EIA cAMP ELISA kit (Assay Design) wereused to indicate anti-LT antibody neutralizing activity. Neutralizinganti-LT antibodies neutralize CT toxin and prevent CT from stimulatingcAMP in T-84 cells, thus resulting in a lower intracellular cAMP level.The serum sample (30 μl; in a final dilution of 1:33.3) pooled from eachgroup of mice immunized with ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’,‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’, co-immunized with ‘CFA/I/II/IV’and ‘3xSTa_(N12S)-LT_(toxoid)’, or the control group was incubated withCT toxin (10 ng, in 150 μl cell culture medium) for 30 minutes at roomtemperature, and the serum-toxin mixture was added to T-84 cells (1 mlof final volume with cell culture medium). Intracellular cAMPconcentration (pmole/ml) in T-84 cells was measured after 3 hourincubation at a CO₂ incubator, with the mean cAMP and standard deviation(from four to six replicates) of each group indicated as columns andbars. The cAMP in T-84 cells incubated with cell culture medium alone(without CT or serum; no CT toxicity), or with CT in culture medium(without serum; CT toxicity) were also measured as references.

FIGS. 17A-17C show nucleic acid sequences of a modification of theCFA-STa_(N12S)-LT_(toxoid) MEFA of the present invention. Thisembodiment comprises 3 copies of the toxoid STa_(N12S) in order tofurther enhance the fusion antigen anti-STa immunogenicity.

FIG. 18A is a schematic showing the CFA-toxoid MEFA carries 3 copies ofSTa_(N12S) at the N terminus, the C-terminus, and after the 192 residueof the LT A subunit gene, the CFA MEFA, and 160 to 192 AAs and 193 to240 AAs of the LT A, and the LT B subunit.

FIG. 18B is a photograph of a Coomassie blue staining gel showed purityof the extracted his-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA proteinto be used to immunize mice and pregnant sow.

FIG. 18C is a photograph of Western blots showing detection of thishis-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA protein by anti-CFA mAbshybridoma supernatant (provided by Dr. AM Svennerholm), and anti-STa andanti-CT rabbit serum. Lane M, the protein marker; lane 1, extractedproteins of 9419; lane 2, total protein of host E. coli BL21.

FIG. 19 is a graph depicting titers of anti-STa antibodies inintraperitoneally immunized mice.

FIG. 20 is a pair of bar graphs showing that induced anti-STa antibodiesin mouse serum can completely neutralize STa toxin.

FIGS. 21A-21H are graphs depicting the double mutant LT(LT_(R192G/L211A); gift from PATH) was found an effective adjuvant toenhance the his-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA in inducingantibody responses to all 7 adhesins and both toxins.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one or more embodiments of the present invention,instead of randomly stacking individual epitopes from each adhesin withlinkers to generate a multiepitope antigen, the CFA/I major subunit wasused CfaB as the backbone, and antigenic prediction software was appliedto embed epitopes of CS1-CS6 (by substituting surface-exposed but lessantigenic CfaB epitopes) so that the resultant multiepitope CFA proteinwould have antigenicity propensity similar to the CfaB subunit. Thismultiepitope CFA protein thereby has a relatively stable structure and abetter presentation for the CS1-CS6 epitopes to induce host immuneresponses. When carried by the CFA/I Cfa operon, this multiepitope CFAprotein can be expressed as a multiepitope CFA adhesin to be used forkilled or live whole-cell vaccine development. This approach, describedherein as an embodiment of the present invention, to constructmultiepitope antigen against multiple virulence factors can be generallyapplied in developing multivalent vaccines against pathogens expressingheterogeneous virulence factors.

Data from the embodiments of the present invention indicate that theseconstructed multiepitope CFA antigens elicited antibodies cross reactiveto CAF/I, CFA/II and CFA/IV adhesins, and that anti-adhesin antibodiesin the serum significantly inhibited adherence of ETEC or E. colistrains expressing these 7 adhesins to Caco-2 or T-84 cells, and showsits application in developing effective ETEC anti-adhesin vaccines. Inconjunction to a toxoid fusion antigen that induces antibodiesneutralizing against both LT and STa toxins, this multiepitope CFAantigens of the present invention can be used to develop broadlyprotective vaccines against ETEC diarrhea.

In accordance with an embodiment, the present invention provides apolypeptide molecule comprising one or more of the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6).

In accordance with another embodiment, the present invention provides apolypeptide molecule comprising one or more of the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6) comprising the amino acid sequence of SEQ ID NO: 1, or having atleast 90% identity to SEQ ID NO: 1.

As used herein the polypeptide antigens used in the fusion molecule ofthe present invention comprise antigenic epitopes of CFA/I(¹⁵⁹SGVVSLVMT¹⁶⁷, SEQ ID NO: 4); CS1 (⁹⁷PTLQIPVS¹⁰⁴, SEQ ID NO: 5); CS2(¹⁶¹LVSIVLT¹⁶⁷, SEQ ID NO: 6); CS3 (⁶¹NTLVGVLTL⁶⁹, SEQ ID NO: 7); CS4(⁷⁹KNVLVKLV⁸⁶, SEQ ID NO: 8); CS5 (⁸³DFFIVPVSG⁹¹, SEQ ID NO: 9); and CS6(⁷²QVTVYPV⁷⁸, SEQ ID NO: 10).

In accordance with another embodiment, a multiepitope CFA subunit genewas genetically fused to a STa-LT_(toxoid) fusion gene (which consistsof a unique single open reading frame to coding a single peptide, as theLT genes were modified for disruption of the cistron gene structure andremoval of signal peptides) for expression of two CFA-toxoidmultiepitope fusion antigens (CFA/I/II/IV-STa_(A14Q)-LT_(toxoid) &CFA/I/II/IV-STa_(N12S)-LT_(toxoid)). The constructed‘CFA/I/II/IV-STa_(toxoid)-LT_(toxoid)’ fusions were examined for antigensafety and immunogenicity in a murine model. Elicited antibodies forneutralization activities were then measured to assess potency of fusionproteins in developing broadly protective vaccines against ETEC strains.Additionally, immune responses were comparatively examined in miceimmunized with CFAI/IV-STa_(toxoid)-LT_(toxoid) fusion with those inmice co-administrated with the CFA multiepitope fusion antigen and aSTa_(toxoid)-LT_(toxoid) fusion in order to further assess applicationof fusion antigens in multivalent vaccine development.

In accordance with an embodiment, the present invention provides a MEFAcomprising a polypeptide molecule encoding the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6) and further comprising heat-labile toxin (LT) and heat-stable toxin(STa).

In accordance with an embodiment, the present invention provides apolypeptide molecule comprising the colonization factor antigens (CFA)antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6) andfurther comprising heat-labile toxin (LT) and heat-stable toxin (STa).

In accordance with another embodiment, the present invention provides apolypeptide molecule comprising the colonization factor antigens (CFA)antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6) andfurther comprising heat-labile toxin (LT) and heat-stable toxin (STa)comprising the amino acid sequence of SEQ ID NO: 2 and/or 3.

As used herein the polypeptide antigens used in the fusion molecule ofthe present invention comprise LTA131-240, (SEQ ID NO: 11); LTB1-100,(SEQ ID NO: 12); STa_(toxoid) with an A14Q mutation (SEQ ID NO: 13), ora N12S mutation (SEQ ID NO: 14).

It will be understood by one of ordinary skill in the art that theantigenic polypeptides or fusion proteins provided herein can includeone or more copies of the antigen polypeptide. For example, the fusionpolypeptide can comprise 1, 2, 3 or more STa toxoid, LTa, CS or CFAfragments or polypeptides.

It will be understood by one of ordinary skill in the art that theantigenic polypeptides or fusion proteins provided herein can includeone or more copies of the following linker peptides LGA, GPVD (SEQ IDNO: 36), Gly-Pro linker GPGP (SEQ ID NO: 37), and L-linker (DPRVLSS, SEQID NO: 39).

In accordance with an embodiment, the present invention provides afusion polypeptide molecule comprising CFA antigens I, II and IV,STa_(toxoid) and LT_(toxoid).

In some embodiments, the present invention provides a fusion polypeptidemolecule comprising CFA antigens I, II and IV, STa_(toxoid) andLT_(toxoid) wherein the STa_(toxoid) has an A14Q mutation or a N12Smutation.

In accordance with an embodiment, the present invention provides apolynucleotide encoding a fusion polypeptide molecule comprising CFAantigens I, II and IV, STa_(toxoid) and LT_(toxoid).

In some embodiments, the present invention provides a polynucleotideencoding a fusion polypeptide molecule comprising CFA antigens I, II andIV, STa_(toxoid) and LT_(toxoid) antigens, wherein the STa_(toxoid) hasan A14Q mutation or a N12S mutation.

In some embodiments the CFA antigens I, II and IV comprise one or moreof CS1, CS2, CS3, CS4, CS5 and CS6 adhesins. In some embodiments, CFAantigens I, II and IV comprise each of CS1, CS2, CS3, CS4, CS5 and CS6adhesins.

In accordance with an embodiment, the present invention provides afusion polypeptide molecule comprising three STa_(toxoid) antigens and aLT_(toxoid) antigen.

In some embodiments, the present invention provides a polynucleotideencoding a fusion polypeptide molecule comprising three STa_(toxoid)antigens and a LT_(toxoid) antigen.

In accordance with an embodiment, the present invention provides afusion polypeptide molecule comprising three STa_(toxoid) antigens and aLT_(toxoid) antigen, wherein the three STa_(toxoid) antigens areselected from the group consisting of STa_(A14Q) and STa_(N12S).

In accordance with an embodiment, the present invention provides apolynucleotide encoding a fusion polypeptide molecule comprising threeSTa_(toxoid) antigens and a LT_(toxoid) antigen, wherein the threeSTa_(toxoid) antigens are selected from the group consisting ofSTa_(A14Q) and STa_(N12S).

In some embodiments, the three STa_(toxoid) antigens are the same.

In accordance with another embodiment, the present invention provides afusion polypeptide molecule comprising CFA antigens I, II and IV, threeSTa_(toxoid) antigens and a LT_(toxoid) antigen, wherein theSTa_(toxoid) has an A14Q mutation or a N12S mutation.

In an embodiment, the present invention provides a fusion polypeptidemolecule comprising CFA antigens I, II and IV, three STa_(toxoid)antigens and a LT_(toxoid) antigen, wherein the STa_(toxoid) has a N12Smutation.

In accordance with another embodiment, the present invention provides apolynucleotide encoding a fusion polypeptide molecule comprising CFAantigens I, II and IV, three STa_(toxoid) antigens and a LT_(toxoid)antigen, wherein the STa_(toxoid) has an A14Q mutation or a N12Smutation.

In an embodiment, the present invention provides a polynucleotideencoding a fusion polypeptide molecule comprising CFA antigens I, II andIV, three STa_(toxoid) antigens and a LT_(toxoid) antigen, wherein theSTa_(toxoid) has a N12S mutation.

In accordance with an embodiment, the present invention provides afusion polypeptide molecule comprising three STa_(toxoid) antigens and aLT_(toxoid) antigen, wherein the three STa_(toxoid) antigens lack ahistidine tag and are selected from the group consisting of STa_(A14Q)and STa_(N12S).

In some embodiments, the present invention provides a fusion polypeptidemolecule comprising three STa_(toxoid) antigens and a LT_(toxoid)antigen, wherein the three STa toxoid antigens lack a histidine tag andhave the N12S mutation.

In accordance with an embodiment, the present invention provides apolynucleotide encoding a fusion polypeptide molecule comprising threeSTa_(toxoid) antigens and a LT_(toxoid) antigen, wherein the threeSTa_(toxoid) antigens lack a histidine tag and are selected from thegroup consisting of STa_(A14Q) and STa_(N12S).

In some embodiments, the present invention provides a polynucleotideencoding a fusion polypeptide molecule comprising three STa_(toxoid)antigens and a LT_(toxoid) antigen, wherein the three STa_(toxoid)antigens lack a histidine tag and have the N12S mutation.

In accordance with another embodiment, the present invention provides afusion polypeptide molecule comprising CFA antigens I, II and IV, threeSTa_(toxoid) antigens lacking a histidine tag and a LT_(toxoid) antigen,wherein the STa_(toxoid) has an A14Q mutation or a N12S mutation.

In an embodiment, the present invention provides a fusion polypeptidemolecule comprising CFA antigens I, II and IV, three STa_(toxoid)antigens lacking a histidine tag and a LT_(toxoid) antigen, wherein theSTa_(toxoid) has a N12S mutation.

It will be understood by those of skill in the art that the fusionpolypeptides disclosed herein can be used to prepare medicaments for usein treatment of various enteric diseases.

In accordance with an embodiment, the present invention provides the useof a polypeptide molecule comprising one or more of the colonizationfactor antigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV(CS4, CS5, CS6) and/or comprising the amino acid sequence of SEQ ID NO:1, or having at least 90% identity to SEQ ID NO: 1 in the treatment offor the therapy or prophylaxis of Enterotoxigenic Escherichia coli(ETEC), infection in a subject or for the therapy or prophylaxis ofdiarrhea in a subject comprising administering to the subject aneffective amount of the polypeptide molecule.

In accordance with a further embodiment, the present invention providesthe use of a polypeptide molecule comprising the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6) and further comprising heat-labile toxin (LT) and heat-stable toxin(STa) and/or comprising the amino acid sequence of SEQ ID NO: 2 and/or3, in the treatment of for the therapy or prophylaxis of EnterotoxigenicEscherichia coli (ETEC), infection in a subject or for the therapy orprophylaxis of diarrhea in a subject comprising administering to thesubject an effective amount of the polypeptide molecule.

In accordance with an embodiment, the present invention provides the useof a fusion polypeptide molecule comprising CFA antigens I, II and IV,STa_(toxoid) and LT_(toxoid), in the treatment of for the therapy orprophylaxis of Enterotoxigenic Escherichia coli (ETEC), infection in asubject or for the therapy or prophylaxis of diarrhea in a subjectcomprising administering to the subject an effective amount of thepolypeptide molecule.

In accordance with another embodiment, the present invention providesthe use of a fusion polypeptide molecule comprising CFA antigens I, IIand IV, STa_(toxoid) and LT_(toxoid), wherein the STa_(toxoid) has anA14Q mutation or a N12S mutation, in the treatment of for the therapy orprophylaxis of Enterotoxigenic Escherichia coli (ETEC), infection in asubject or for the therapy or prophylaxis of diarrhea in a subjectcomprising administering to the subject an effective amount of thepolypeptide molecule.

In accordance with an embodiment, the present invention provides the usea fusion polypeptide molecule comprising CFA antigens I, II and IV,three STa_(toxoid) antigens and a LT_(toxoid) antigen, wherein theSTa_(toxoid) has an A14Q mutation or a N12S mutation, in the treatmentof for the therapy or prophylaxis of Enterotoxigenic Escherichia coli(ETEC), infection in a subject or for the therapy or prophylaxis ofdiarrhea in a subject comprising administering to the subject aneffective amount of the polypeptide molecule.

As used herein, the term “multiepitope fusion antigen (MEFA)” is anapproach which combines both the fusion and epitope vaccine strategies.MEFAs can be made and developed for production of multivalent vaccinesagainst other heterogeneous pathogens or perhaps different diseases. TheMEFA methods of the present invention allows for the addition of anyantigenic element, such as future-identified virulence factors (oremerging factors that become more prevalent or virulent, such as CS21adhesin, which shows association with traveler's diarrhea), or multiplecopies of one element such as STa toxoid.

The term, “amino acid” includes the residues of the natural α-aminoacids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Ile, Leu,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well as-amino acids, synthetic and unnatural amino acids. Many types of aminoacid residues are useful in the adipokine polypeptides and the inventionis not limited to natural, genetically-encoded amino acids. Examples ofamino acids that can be utilized in the peptides described herein can befound, for example, in Fasman, 1989, CRC Practical Handbook ofBiochemistry and Molecular Biology, CRC Press, Inc., and the referencecited therein. Another source of a wide array of amino acid residues isprovided by the website of RSP Amino Acids LLC.

The term, “peptide,” as used herein, includes a sequence of from four tosixteen amino acid residues in which the α-carboxyl group of one aminoacid is joined by an amide bond to the main chain (α- or β-) amino groupof the adjacent amino acid. The peptides provided herein for use in thedescribed and claimed methods and compositions can be cyclic.

In accordance with an embodiment, the present invention provides avaccine composition comprising the polypeptide molecule describedherein, and a pharmaceutically acceptable carrier.

In accordance with another embodiment, the present invention provides amethod for the therapy or prophylaxis of Enterotoxigenic Escherichiacoli (ETEC), infection in a subject comprising administering to thesubject the polypeptide molecules or the vaccines described herein.

In accordance with a further embodiment, the present invention providesa method for the therapy or prophylaxis of diarrhea in a subjectcomprising administering to the subject the polypeptide molecules or thevaccines described herein.

In accordance with still another embodiment, the present inventionprovides a method for the therapy or prophylaxis of EnterotoxigenicEscherichia coli (ETEC), infection in a subject comprising administeringto the subject the polypeptide molecules or the vaccines describedherein and administration of at least one additional therapeutic agent.

The term, “amount effective to treat diarrhea” is that amount effectiveto treat, ameliorate, or prevent acute and prolonged (<1 month) orchronic (>1 month) diarrhea or symptoms thereof, or to exhibit adetectable therapeutic or preventative effect.

The precise effective amount for a human subject will depend upon theseverity of the subject's disease state, general health, age, weight,gender, diet, time and frequency of administration, drug combination(s),reaction sensitivities, and tolerance or response to therapy. A routineexperimentation can determine this amount and is within the judgment ofthe medical professional. Compositions may be administered individuallyto a patient, or they may be administered in combination with otherdrugs, hormones, agents, and the like.

With respect to peptide compositions described herein, the carrier canbe any of those conventionally used, and is limited only byphysico-chemical considerations, such as solubility and lack ofreactivity with the active compound(s), and by the route ofadministration. The carriers described herein, for example, vehicles,adjuvants, excipients, and diluents, are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe carrier be one which is chemically inert to the active agent(s), andone which has little or no detrimental side effects or toxicity underthe conditions of use. Examples of the carriers include soluble carrierssuch as known buffers which can be physiologically acceptable (e.g.,phosphate buffer) as well as solid compositions such as solid-statecarriers or latex beads.

The carriers or diluents used herein may be solid carriers or diluentsfor solid formulations, liquid carriers or diluents for liquidformulations, or mixtures thereof.

Solid carriers or diluents include, but are not limited to, gums,starches (e.g., corn starch, pregelatinized starch), sugars (e.g.,lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g.,microcrystalline cellulose), acrylates (e.g., polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

For liquid formulations, pharmaceutically acceptable carriers may be,for example, aqueous or non-aqueous solutions, or suspensions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol, andinjectable organic esters such as ethyl oleate. Aqueous carriersinclude, for example, water, alcoholic/aqueous solutions, cyclodextrins,emulsions or suspensions, including saline and buffered media.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include, for example, sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's andfixed oils. Formulations suitable for parenteral administration include,for example, aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives.

It will be appreciated by one of skill in the art that, in addition tothe above-described vaccine compositions, the polypeptides of theinvention can be formulated as inclusion complexes, such as cyclodextrininclusion complexes, or liposomes.

Intravenous vehicles include, for example, fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Examples are sterile liquids such as water andoils, with or without the addition of a surfactant and otherpharmaceutically acceptable adjuvants. In general, water, saline,aqueous dextrose and related sugar solutions, and glycols such aspropylene glycols or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

In one or more preferred embodiments, the route of administration of theabove-described vaccine compositions, the route is intradermal orsubcutaneous for polypeptide vaccine delivery and in other embodiments,the route is oral for whole cell vaccine.

In addition, in an embodiment, the compositions comprising polypeptidesor vaccines thereof, may further comprise binders (e.g., acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g., cornstarch, potato starch, alginic acid, silicon dioxide,croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCl., acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., cremophor, glycerol,polyethylene glycerol, benzlkonium chloride, benzyl benzoate,cyclodextrins, sorbitan esters, stearic acids), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite, butylated hydroxyanisole),stabilizers (e.g., hydroxypropyl cellulose, hyroxypropylmethylcellulose), viscosity increasing agents (e.g., carbomer, colloidalsilicon dioxide, ethyl cellulose, guar gum), sweetners (e.g., aspartame,citric acid), preservatives (e.g., thimerosal, benzyl alcohol,parabens), lubricants (e.g., stearic acid, magnesium stearate,polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidalsilicon dioxide), plasticizers (e.g., diethyl phthalate, triethylcitrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodiumlauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines),coating and film forming agents (e.g., ethyl cellulose, acrylates,polymethacrylates), and/or adjuvants.

The choice of carrier will be determined, in part, by the particularpeptide containing compositions, as well as by the particular methodused to administer the composition. Accordingly, there are a variety ofsuitable formulations of the pharmaceutical compositions of theinvention. The following formulations for parenteral, subcutaneous,intravenous, intramuscular, intraarterial, intrathecal andinterperitoneal administration are exemplary, and are in no waylimiting. More than one route can be used to administer the compositionsof the present invention, and in certain instances, a particular routecan provide a more immediate and more effective response than anotherroute.

As used herein the term “pharmaceutically active compound” or“therapeutically active compound” means a compound useful for thetreatment or modulation of a disease or condition in a subject sufferingtherefrom. Examples of pharmaceutically active compounds can include anydrugs known in the art for treatment of disease indications.

Other therapeutically active compounds included in the pharmaceuticalcompositions suitable for use in the methods of the present inventioninclude antidiarrheal agents. Examples of such agents include, but arenot limited to: bulking agents like methylcellulose, guar gum, kaolinsuspensions or plant fiber (bran, sterculia, isabgol, etc.) are used fordiarrhea in functional bowel disease and to control ileostomy output;absorbents which absorb toxic substances that cause infective diarrhea,such as methylcellulose; anti-inflammatory solutions, such as bismuthsubsalicylate; and opioids, such as loperamide and diphenoxylate.

For purposes of the present invention, the term “diarrhea,” as usedherein means frequent, poorly formed, loose, watery stools of a subject.A subject having diarrhea means the subject is passing loose stools atleast three times a day. The term “acute diarrhea” is a common problemthat usually lasts <7 days but can last in a protracted or prolongedform for <21 days. Diarrhea lasting more than 2 days is often a sign ofan enteropathogenic infection. The term “chronic diarrhea” meansdiarrhea that lasts at least 4 weeks. Chronic diarrhea symptoms may becontinual or intermittent. The term “traveler's diarrhea” meansdiarrheal symptoms associated with travel-related infection. In additionto diarrhea, symptoms may include nausea, vomiting, abdominal pain,fever, sweats, chills, headache, and malaise. Diarrhea may also be theresult of food borne enteropathogens.

Diarrhea of any duration may cause dehydration, which means the bodylacks enough fluid and electrolytes—chemicals in salts, includingsodium, potassium, and chloride—to function properly. Loose stoolscontain more water and electrolytes and often weigh more than solidstools.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof diarrhea in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the disease, e.g., diarrhea, beingtreated or prevented. Also, for purposes herein, “prevention” canencompass delaying the onset of the disease, or a symptom or conditionthereof.

In accordance with an embodiment of the present invention, themedicament for treating a disease in a subject can encompass manydifferent formulations known in the pharmaceutical arts, including, forexample, intravenous and sustained release formulations.

As used herein, the term “treat,” as well as words stemming therefrom,includes diagnostic and preventative as well as disorder remitativetreatment.

As used herein, the term “subject” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

An effective amount of the polypeptides or vaccine compositions, to beemployed therapeutically will depend, for example, upon the therapeuticand treatment objectives, the route of administration, the age,condition, and body mass of the subject undergoing treatment or therapy,and auxiliary or adjuvant therapies being provided to the subject.Accordingly, it will be necessary and routine for the practitioner totiter the dosage and modify the route of administration, as required, toobtain the optimal therapeutic effect. A typical daily dosage mightrange from about 0.1 mg/kg to up to about 100 mg/kg or more, preferablyfrom about 0.1 to about 10 mg/kg/day depending on the above-mentionedfactors. Typically, the clinician will administer antibody until adosage is reached that achieves the desired effect. In accordance withsome embodiments, the dosage range for intradermal vaccine can be about1 to 500 μg, preferably about 30 to 40 g per dose in adults.

Included in the scope of the invention are functional variants of theinventive polypeptides or vaccine compositions described herein. Theterm “functional variant” as used herein refers polypeptides or vaccinecompositions having substantial or significant sequence identity orsimilarity to polypeptides or vaccine compositions, which functionalvariant retains the biological activity of polypeptides or vaccinecompositions of which it is a variant. In reference to the parentpolypeptide, or protein, the functional variant can, for instance, be atleast about 30%, 50%, 75%, 80%, 90%, 98% or more identical in amino acidsequence to the parent polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent polypeptides, or proteins of the presentinvention with at least one conservative amino acid substitution.Conservative amino acid substitutions are known in the art, and includeamino acid substitutions in which one amino acid having certain physicaland/or chemical properties is exchanged for another amino acid that hasthe same chemical or physical properties. For instance, the conservativeamino acid substitution can be an acidic amino acid substituted foranother acidic amino acid (e.g., Asp or Glu), an amino acid with anonpolar side chain substituted for another amino acid with a nonpolarside chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val,etc.), a basic amino acid substituted for another basic amino acid (Lys,Arg, etc.), an amino acid with a polar side chain substituted foranother amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr,Tyr, etc.), etc.

Functional variants can also include extensions of the inventivepolypeptides. For example, a functional variant of the inventivepolypeptides can include 1, 2, 3, 4 and 5 additional amino acids fromeither the N-terminal or C-terminal end of the polypeptide.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the inventive polypeptides, or proteins with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the inventive polypeptides, or proteins.

The inventive polypeptides or proteins can consist essentially of thespecified amino acid sequence or sequences described herein, such thatother components of the functional variant, e.g., other amino acids, donot materially change the biological activity of the functional variant.

The inventive polypeptides or proteins (including functional portionsand functional variants) of the invention can comprise synthetic aminoacids in place of one or more naturally-occurring amino acids. Suchsynthetic amino acids are known in the art, and include, for example,aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid,homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbomane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The inventive polypeptides or proteins (including functional portionsand functional variants) can be glycosylated, amidated, carboxylated,phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfidebridge, or converted into an acid addition salt and/or optionallydimerized or polymerized, or conjugated.

The inventive polypeptides or proteins (including functional portionsand functional variants thereof) can be obtained by methods known in theart. Suitable methods of de novo synthesizing polypeptides and proteinsare described in references, such as Chan et al., Fmoc Solid PhasePeptide Synthesis, Oxford University Press, Oxford, United Kingdom,2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,Inc., 2000; Epitope Mapping, ed. Westwoood et al., Oxford UniversityPress, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also,polypeptides and proteins can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, N Y, 1994. Further, some ofthe ICRX-CP s, polypeptides, and proteins of the invention (includingfunctional portions and functional variants thereof) can be isolatedand/or purified from a source, such as a plant, a bacterium, an insect,a mammal, e.g., a rat, a human, etc. Methods of isolation andpurification are well-known in the art. Alternatively, the inventivepolypeptides or proteins described herein (including functional portionsand functional variants thereof) can be commercially synthesized bycompanies, such as Synpep (Dublin, Calif.), Peptide Technologies Corp.(Gaithersburg, Md.), and Multiple Peptide Systems (San Diego, Calif.).In this respect, the inventive TCRs, polypeptides, and proteins can besynthetic, recombinant, isolated, and/or purified.

Further provided by the invention are nucleic acid molecules comprisinga nucleotide sequence encoding the inventive polypeptides or proteinsdescribed herein (including functional portions and functional variantsthereof).

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered intemucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g., hosphorothioatederivatives and acridine substituted nucleotides). Examples of modifiednucleotides that can be used to generate the nucleic acids include, butare not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The invention also provides substituted nucleic acid sequences whichencode any of the substituted inventive polypeptides, substitutedpolypeptides, substituted proteins, or substituted functional portionsor functional variants thereof.

In some embodiments, the substituted nucleic acid sequence may beoptimized. Without being bound to a particular theory, it is believedthat optimization of the nucleic acid sequence increases the translationefficiency of the mRNA transcripts. Optimization of the nucleic acidsequence may involve substituting a native codon for another codon thatencodes the same amino acid, but can be translated by tRNA that is morereadily available within a cell, thus increasing translation efficiency.Optimization of the nucleic acid sequence may also reduce secondary mRNAstructures that would interfere with translation, thus increasingtranslation efficiency.

The polynucleotide sequence encoding an inventive polypeptide of theinvention includes the exemplified sequences, as well as conservativevariations of the exemplified polypeptide sequences. The term“conservative variation” as used herein refers to a replacement of anamino acid residue by another, biologically similar amino acid residue.Examples of conservative variations include the substitution of ahydrophobic residue such as isoleucine, valine, leucine or methioninefor another, or the substitution of a polar residue for another, such asthe substitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine, and the like. The term “conservativevariation” also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that an antibody thatspecifically interacts with the substituted polypeptide also isspecifically immunoreactive with the unsubstituted polypeptide.

A polynucleotide of the invention can be obtained by several methods.For example, the polynucleotide can be isolated using hybridization orcomputer-based techniques which are well known in the art. Theseinclude, but are not limited to: 1) hybridization of genomic or cDNAlibraries with probes to detect homologous nucleotide sequences; 2)antibody screening of expression libraries to detect cloned DNAfragments with shared structural features; 3) polymerase chain reaction(PCR) on genomic DNA or cDNA using primers capable of annealing to theDNA sequence of interest; 4) computer searches of sequence databases forsimilar sequences; and 5) differential screening of a subtracted DNAlibrary.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or moreinventive polypeptides described herein are provided. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more inventive polypeptides or fragments thereof describedherein) in a form suitable for expression of the nucleic acid sequencein a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence encoding one or more inventive polypeptides is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cells and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences, e.g., adipose tissue). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors described herein can be introduced into host cells to therebyproduce proteins or portions thereof, including fusion proteins orportions thereof, encoded by nucleic acids as described herein (e.g.,inventive peptides).

In accordance with an embodiment, the present invention provides the useof a polypeptide molecule comprising one or more of the colonizationfactor antigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV(CS4, CS5, CS6) and/or comprising the amino acid sequence of SEQ ID NO:1, or having at least 90% identity to SEQ ID NO: 1 in the treatment offor the therapy or prophylaxis of Enterotoxigenic Escherichia coli(ETEC), infection in a subject or for the therapy or prophylaxis ofdiarrhea in a subject comprising administering to the subject aneffective amount of the polypeptide molecule.

In accordance with another embodiment, the present invention providesthe use of a polypeptide molecule comprising the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6) and further comprising heat-labile toxin (LT) and heat-stable toxin(STa) and/or comprising the amino acid sequence of SEQ ID NO: 2 and/or3, in the treatment of for the therapy or prophylaxis of EnterotoxigenicEscherichia coli (ETEC), infection in a subject or for the therapy orprophylaxis of diarrhea in a subject comprising administering to thesubject an effective amount of the polypeptide molecule.

In accordance with a further embodiment, the present invention providesthe use of a fusion polypeptide molecule comprising CFA antigens I, IIand IV, STa_(toxoid) and LT_(toxoid), in the treatment of for thetherapy or prophylaxis of Enterotoxigenic Escherichia coli (ETEC),infection in a subject or for the therapy or prophylaxis of diarrhea ina subject comprising administering to the subject an effective amount ofthe polypeptide molecule.

In some embodiments, the fusion polypeptides of the present inventionare administered to the subject in a vaccine formulation.

In some embodiments, the administration of the fusion polypeptides ofthe present invention can be combined with the administration of atleast one additional therapeutic agent.

It will be understood that the additional therapeutic agents which canbe combined with administration of the fusion polypeptides of thepresent invention can include other drugs or biological agents. Forexample, the additional therapeutic agents can include anti-motility,antidiarrheals, antibiotics, antihelminths, and anti-parasiticals.

EXAMPLES

Bacterial strains and plasmids. E. coli strains and plasmids used inthis study are listed in Table 1. ETEC field isolates deposited at JohnsHopkins University and the E. coli Reference Strain Center at Universityof Gothenburg (Sweden), and recombinant CS1 and CS2 E. coli strains(gifts from Dr. J. Scott at Emory University) (Infect Immun58(11):3594-3600 (1990); Infect Immun 63(12):4849-4856 (1995)) were usedfor CFA adhesin extraction and in antibody adherence inhibition assays.E. coli BL21 (GE Healthcare, Piscataway, N.J.) and vector pET28α(Novagen, Madison, Wis.) were used to express the multiepitope CFAprotein.

Recombinant strains 9175 (CFA/I/II/IV), 9164(3xSTa_(A14Q)-tmLT_(S63K/R192G/L211A)), and 9318(3xSTa_(N12S)-LT_(toxoid)) were used as templates forCFA/I/II/IV-STa-toxoid-LT_(toxoid) fusion construction. E. coli BL21 (GEHealthcare, Piscataway, N.J.) and vector pET28α (Novagen, Madison, Wis.)were used as templates first to construct theCFA/I/II/IV-STa_(A14Q)-dmLT MEFA gene. Recombinant strain 9318(3xSTa_(N12S)-dmLT_(R192G/L211A)) was included for theCFA/I/II/IV-STa_(N12S)-dmLT MEFA after 3xSTa_(N12S)-LT_(toxoid) wasidentified as the optimal toxoid fusion in inducing anti-STa antibodyresponse. CFA multiepitope fusion antigen (MEFA) construction.

Epitopes from each of the CFA/I, CS1-CS6 major structural subunits(CfaB, CooA, CotA, CstH, CsaB, CsfA and CssA) were predicted withweb-based programs (Bioinformatics 14(10):892-893 (1998); J Mol Biol225(2):487-494 (1992); J Mol Recognit 16(1):20-22 (2003); Methods MolBiol 409 (2007)) and classic algorithims (Proc Natl Acad Sci USA78(6):3824-3828 (1981); Biochemistry 17(20):4277-4285 (1978); Journal oftheoretical biology 21(2):170-201 (1968)). Antigenic epitopes predictedby all or a majority of the programs were initially selected, and CFA/Imajor structural subunit CfaB was used as the backbone for constructingthe CFA multiepitope fusion antigen. With nucleotides coding the mostantigenic epitope were retained, the CfaB gene (cfaB) had nucleotidescoding the surface-exposed but less antigenic epitopes substituted withnucleotides coding the most antigenic epitope of the CS1-CS6 majorstructural subunits, in a sequence so that the protein encoded by thischimeric gene is similar to CfaB in antigenicity propensity. Thischimeric gene, after silent mutation to fit PCR primer design andcloning purposes, was synthesized (Integrated DNA Technologies, Inc.,Coralville, Iowa). The synthesized gene was amplified in a PCR with pfuTaq polymerase (Strategene, La Jolla, Calif.) and primers pETCFA-F(5′-gtgagtgctagcgcagtagaggattttttcatt-′3; NheI site underlined) (SEQ IDNO: 15) and pETCFA-R (5′-ctctcggccgttatcaggctcccaaagtcattacaag-′3; EagIsite underlined) (SEQ ID NO: 16), digested with NheI/EagI restrictionenzymes (New England BioLabs, Ipswich, Mass.), and ligated intoexpression vector pET28a using standard protocols. The clonedmultiepitope CFA subunit gene was verified with DNA sequencing.

Multiepitope ‘CFA/I/II/IV-STa_(toxoid)-LT_(toxoid)’ fusion construction.Splicing overlap extension (SOE) PCR was used to construct geneticfusions as described previously. Two PCR products, the multiepitope CFAcarrying antigenic epitopes of 7 CFA adhesins (CFA/I, CFA/II, andCFA/IV) and the STa-LT_(toxoid) fusion including 2 copies of theSTa-toxoid, LTA(131-240) and LTB were overlapped for theCFA/I/II/IV-STa_(toxoid)-LT_(toxoid) chimeric gene. The multiepitope CFAfragment was amplified with primers T7-F (5′-taatacgactcactataggg-′3)(SEQ ID NO: 17) and CFA-toxoid-R(5′-accaaaggctcccaaagtcattacaagagatactactcctga-′3) (SEQ ID NO: 18) usingplasmid pCFA/I/II/IV as the DNA template. Two toxoid fragments wereamplified with primers CFA-toxoid-F(5′-gtaatgactttgggagcctttggtgtgattgatgaacgattacatcgt-′3) (SEQ ID NO: 19)and T7-R (5′-tgctagttattggtcaggggt-′3) (SEQ ID NO: 20) using plasmidp3xSTa_(A14Q)-tmLT and plasmid p3xSTa_(N12S)-dmLT respectively as theDNA templates. Primers hSTa_(N12S)-F (5′-gaa ttg tgt tgt age cct gettgt-′3) (SEQ ID NO: 32) and hSTa_(N12S)-R (5′-aca agc agg get aca acacaa ttc-′3) (SEQ ID NO: 33); and hSTa_(A14Q)-F (tgt tgt aat cct cag tgtacc ggg-′3) (SEQ ID NO: 34) and hSTa_(A14Q)-R (5′-ccc ggt aca ctg aggatt aca aca-′3) (SEQ ID NO: 35) were used to mutate the STa gene for STatoxoids STa_(N12S) and STa_(A14Q) respectively. Each SOE product wasfurther amplified with T7-F and T7-R primers, digested with NheI/EagIrestriction enzymes (New England BioLabs, Ipswich, Mass.), ligated intoexpression vector pET28α, and verified with DNA sequencing.

Expression and detection of the CFA MEFA protein. E. coli strain BL21was transformed with the plasmid carryingCFA/I/II/IV-STa_(A14Q)-LT_(toxoid) or CFA/I/II/IV-STa_(N12S)-LT_(toxoid)chimeric gene to express two CFA/I/II/IV-STa_(toxoid)-LT_(toxoid) MEFAproteins. This strain was grown in 5 ml Luria Bertani (LB) brothsupplemented with kanamycin (30 μg/ml) at 37° C. overnight on a shaker(150 rpm). Overnight growth was added to 500 ml 2× YT (2× Yeast extractand Tryptone) medium for continuous incubation until the optical densityreached 0.5 at 600 nm (OD600). The culture was then induced withisopropyl-1-thio-β-D-galactoside (IPTG; 0.5 mM) and incubated for 4 morehours. The bacterial culture was centrifuged at 5,000×g for 20 minutes,and pellets were suspended into 10 ml bacterial protein extractionreagent (B-PER, in phosphate buffer; Pierce, Rockford, Ill.) for totalinsoluble protein (inclusion body fraction) extraction.

Recombinant 6×His-tagged CFA multiepitope fusion antigen protein wasfurther extracted from total insoluble protein extracts (in denaturedbuffer) to a purity of greater than 90% with Ni-nitrilotriacetic acid(NTA) agarose (QIAGEN, Valencia, Calif.). Extracted 6×His-tagged proteinwas refolded using a Protein Refolding kit by following themanufacturer's protocol (Novagen, Madison, Wis.), dialyzed in 20 mMTris-HCl buffer overnight at 4° C., and was concentrated (to 1-2 mg/ml)using Spectra/Por® molecular porous membrane tubing (SpectrumLaboratories Inc., Rancho Dominquez, Calif.) and polyethylene glycolcompound (PEG; Sigma, St. Louis, Mo.) (Infection and immunity78(1):316-325 (2010); Clin Vaccine Immunol 18(10):1593-1599 (2011)).

Ten microliters of refolded protein (10-20 μg) was analyzed in 12%sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)with anti-CFA/I (000419; diluted in 1:100), -CS1 (990505; 1:200), -CS2(990505; 1:100), -CS3 (960607; 1:200), -CS4 (000419; 1:100), -CS5(960607; 1:100), and anti-CS6 (060424; 1:200) MAbs hybridoma supernatant(provided by Dr. A. M. Svennerholm), respectively. Rabbit anti-CT serum(1:3300; Sigma) and protein-A column-purified rabbit anti-STa serum(1:3300; provided by Dr. DC Robertson) (Infect. Immun., 78: 316-325(2010)) were used as the primary antibody. IRDye-labeled goat anti-mouseIgG (1:5000; LI-COR, Lincoln, Nebr.) was used as the secondary antibody.Bound CFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) fusion proteins weredetected using a LI-COR Odyssey premium infrared gel imaging system(LI-COR).

Mouse immunization with the CFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) MEFAproteins. Mouse immunization studies complied with the Animal WelfareAct by following the 1996 National Research Council guidelines and wereapproved and supervised by a state veterinarian and by the Kansas StateUniversity Institutional Animal Care and Use Committee.CFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) MEFA proteins were verifiednon-toxic using T-84 cells and EIA cAMP and cGMP kits (Assay Design, MI)prior to being used in mouse immunization as previously described. Fourgroups of 6- to 8-week-old female BALB/c mice (Charles RiverLaboratories International, Inc., Wilmington, Mass.) were included inthe immunization study. The first group of 8 mice was each injectedintraperitoneally (i.p.) with 200 μg CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)refolded protein (in 200 μl 20 mM Tris-HCl) and an equal volume ofFreund's complete adjuvant (CFA; Sigma). The second group of 15 mice waseach injected (i.p.) with 200 μg CFA/I/II/IV-STa_(N12S)-LT_(toxoid)refolded protein (in 200 μl 20 mM Tris-HCl) with 200 μl CFA. The thirdgroup of 16 mice was each co-administered (i.p.) with refoldedCFA/I/II/IV MEFA and toxoid fusion 3xSTa_(N12S)-LT_(toxoid) producedpreviously. To have the molecule copy numbers of CFA/I/II/IV and3xSTa_(N12S)-dmLT antigens be equivalent to 200 μgCFA/I/II/IV-STa_(N12S)-LT_(toxoid) (based on peptide lengths of theCFAI/II/IV and the STa_(N12S)-dmLT peptides), 80 μg CFA/I/II/IV and 150μg 3xSTa_(N12S)-LT_(toxoid) (in a total of 200 μl 20 mM Tris-HCl) with200 μl CFA were injected to each mouse in this co-administration group.The fourth group of 9 mice was each injected (i.p.) with 200 μl CFA and200 μl 20 mM Tris-HCl and served as the control. Two booster injectionsat the same doses as the primary but with Freund's incomplete adjuvant(IFA) were followed at a bi-week interval. In addition, a group of fivemice was each immunized (i.p.) with 200 μg CFA/I fimbria heat-extractedfrom ETEC H10407 and 200 μl Freund's adjuvants (CFA in the primary andIFA in boosters) to serve as a reference for mouse anti-CFA/I antibodyresponse.

Blood and fecal pellets were collected from each mouse prior toimmunization and 10 to 12 days after each immunization. Fecal pelletswere suspended in fecal reconstitution buffer supplemented with proteaseinhibitor phenylmethylsulfonyl fluoride (Sigma) at a ratio of 1:6 (1gram feces to 5 ml buffer), and centrifuged to collect supernatantsreferred as fecal suspension samples. In addition, intestines collectedfrom each mouse at necropsy were minced with a pair of surgical scissorsin fecal reconstitution buffer (1 g intestine tissue in 2.5 ml buffer, a1:3.5 dilution), vortexed for 1 minute, and centrifuged at 10,000×g for10 minutes to collect supernatants as intestinal wash samples. Serumsamples were stored at -80° C. until use. On day 37 after the primary,mice were anesthetized with CO₂ and exsanguinated. Collected serum,fecal suspension and intestine wash samples were stored at −80° C. untiluse.

Mouse anti-CFA antibody titration. Anti-CFA/I, -CS1, -CS2, -CS3,-CS4/CS6, -CS5/CS6, and anti-CS6 IgG in serum and IgA antibodies infecal and intestine suspension were examined in ELISAs. CFA/I, CS1, CS2,CS3, CS4/CS6, and CS5/CS6 adhesins extracted from ETEC field strains orrecombinant E. coli strains listed in Table 1, in an adhesin heatextraction method described previously (Clin Vaccine Immunol17(12):1859-1867 (2010)), were used as coating antigens. A halfmicrogram of each adhesin [in 100 μl antigen coating buffer (0.015 MNa₂CO₃, 0.035 M NaHCO₃, pH 9.6)] was added to each well of an Immulon2HB plate (Thermo Scientific, Rochester, N.Y.), and incubated for 1 hourat 37° C. then followed by overnight at 4° C. In addition, 100 ngextracted CS6 adhesin (provided by Walter Reed Army Institute ofResearch) was also used to verify anti-CS6 specific antibodies. Coatedplates were washed 3 times with PBST (0.05% Tween-20), blocked with 200μl 10% non-fat milk-PBST for 1 hour at 37° C. After 3 washes with PBST,each well was incubated with serum (1:200, diluted in 2.5% milk-PBST),fecal suspension (1:20 or 1:10) or the intestine suspension sample (1:20or 1:10) of each immunized or control mouse (in triplicate) for 1 hourat 37° C. Plates were washed 5 times with PBST, and each well was addedwith 100 μl 1:3000 diluted HRP-conjugated goat anti-mouse IgG (Sigma) or1:1000 diluted IgA (Sigma) 1 hour at 37° C., followed by 3 more washesand incubation with 100 μl TMB Microwell Peroxidase Substrate System(2-C) (KPL, Gaitherburg, Md.) for 30 minutes at room temperature.Optical density (OD) was measured at a plate reader with 405 nmwavelength. OD readings, greater than 0.3 after subtraction tobackground readings, or the highest readings for the control, werecalculated to antibody titers at a log₁₀ scale.

Mouse anti-CFA and anti-toxin antibody titration. Anti-CFA/I, -CS1,-CS2, -CS3, -CS4/CS6, -CS5/CS6, —STa, and anti-LT IgG antibodies inserum sample of each mouse were titrated as described previously.Briefly, 500 ng CFA/I, CS1, CS2, CS3, CS4/CS6, and CS5/CS6 CFA adhesinsextracted from ETEC field isolates or recombinant E. coli strains (Table1), 10 ng STa-ovalbumin conjugates, or 100 ng LT (List BiologicalLaboratories, Inc., Campbell, Calif.) were coated to each well of H2Bplates (Thermo Scientific, Rochester, N.Y.; for anti-CFA and anti-LTIgG) or Costar plates (Corning Inc., Corning, N.Y.; for anti-STa IgG) totitrate anti-CFA and antitoxin antibodies, respectively. HRP-conjugatedgoat anti-mouse IgG (1:3300; Sigma) and TMB Microwell PeroxidaseSubstrate System (2-C) (KPL, Gaitherburg, Md.) were used to measureoptical density (OD) at a wavelength of 405 nm. OD readings, greaterthan 0.3 after subtraction to background readings were calculated toantibody titers and expressed in a log₁₀ scale.

Anti-adhesin antibody adherence inhibition assay. Mouse serum and fecalsuspension samples were examined for adherence inhibition against ETECstrains expressing CFA/I, CS3, CS4/CS6, or CS5/CS6 adhesins, and E. colirecombinant strains expressing CS1 or CS2, using Caco-2 cells (ATCC,#HTB-37TM) as previously described. Caco-2 cells (7×10⁵) were seeded ateach well of a 12-well tissue culture plate containing DMEM-20% FBS(Fisher Thermo Scientific, Pittsburgh, Pa.). ETEC and E. colirecombinant bacteria grown overnight at 37° C. on sheep blood agarplates were scraped off with Q-tips, and were gently suspended insterile PBS. One hundred microliter of each bacterial suspension(3.5×10⁶ CFUs; with a multiplicity-of-infection ratio set at fivebacteria to one Caco-2 cell) was incubated with 20 μl serum sample whichwas pooled from each group on a shaker (50 rpm) for 1 hour at roomtemperature. The mixture of bacteria and the serum sample was brought to300 μl with PBS, added to each well containing the Caco-2 cells (in 700μl cell culture medium), and incubated 1 hour at 37° C. in a CO₂incubator (5% CO₂). Wells were washed with PBS to remove non-adherentbacteria, and incubated with 0.25% trypsin (200 μl per well) 30 minutesat 37° C. in a CO₂ incubator to dislodge Caco-2 cells. Dislodged Caco-2cells (with adherent bacteria) were collected by centrifugation (15,000g for 10 minutes) and suspended in 1 ml PBS. Suspension was seriallydiluted, plated on LB plates and cultured at 37° C. to count overnightgrown bacteria (CFUs).

Antitoxin antibody neutralization against STa toxin and CT. Serumsamples pooled from mice in each group were also examined for in vitroantibody neutralization activities against STa and CT using EIA cAMP andcGMP kits (Assay Design) and T-84 cells. STa stimulates an increase ofintracellular cyclic GMP levels and CT elevates intracellular cAMPlevels in T-84 cells. Neutralizing antitoxin antibodies neutralizeenterotoxicity thus prevent STa and CT from stimulating intracellularcGMP or cAMP. Therefore, by incubating serum with the toxin, adding themixture to T-84 cells, and measuring cGMP or cAMP levels in the cells,we are able to evaluate neutralization activities of mouse serum IgGantibodies against STa or CT. Serum sample, 30 μl pooled from eachgroup, was incubated with 2 ng STa toxin or 10 ng CT (CT is an homologueof LT, and is commonly used in anti-LT antibody neutralization assay)for 30 minutes at room temperature. The serum and toxin mixture was thenbrought to 300 μl in total with DMEM/F12 medium, and was added to T-84cells (with 700 μl culture medium), and incubated 1 hour at 37° C. in aCO₂ incubator. Cells were washed with PBS and lysed with 300 μl 0.1M HCl(with 0.5% Triton X-100) at room temperature for 20-30 minutes. Afterincubation in a CO₂ incubator for 1 hour (for STa to measure cGMP) or 3h (for CT to measure cAMP), intracellular cGMP or cAMP levels (pmole/ml)in T-84 cell were measured with EIA cGMP or cAMP kit by following themanufacturer's protocol (Assay Design). STa or CT alone (without serum)was used as the control to show enterotoxicity in stimulation of cGMP orcAMP in T-84 cells, and culture medium only (without toxin or serum) wasused to show a baseline of T-84 intracellular cAMP or cGMP level.

Statistical analysis. Data were analyzed by using SAS for windows,version 8 (SAS Institute, Cary, N.C.). Results were expressed as meansstandard deviations. Student's t-test was used to compare the differenttreatment groups. Calculated p values of less than 0.05 were regarded assignificant when treatments were compared at two-tailed distribution andtwo-sample equal or unequal variance.

Example 1

Constructed chimeric protein carried representative epitopes from all 7adhesins. The CFA/I and CS1-CS6 major subunits: CfaB, CooA, CotA, CstH,CsaB, CsfA and CssA, deducted from the cfaB gene sequence (GenBankaccession #M55661), cooA gene (X62495), cotA gene (Z47800.1), cstH gene(M35657.1), csaB gene (AY281092.1), csfA gene (AJ224079.2) and the cssAgene (U04846.1), had peptides ¹⁵⁹SGVVSLVMT¹⁶⁷ (SEQ ID NO: 4),⁹⁷PTLQIPVS¹⁰⁴ (SEQ ID NO: 5), ¹⁶¹LVSIVLT¹⁶⁷ (SEQ ID NO: 6),⁶¹NTLVGVLTL⁶⁹ (SEQ ID NO: 7), ⁷⁹KNVLVKLV⁸⁶ (SEQ ID NO: 8), ⁸³DFFIVPVSG⁹¹(SEQ ID NO: 9), and ⁷²QVTVYPV⁷⁸ (SEQ ID NO: 10), predicted,respectively. By replacing the less antigenic peptides ²⁶KNITVTASV³⁴(SEQ ID NO: 21), ⁶³FESYRVMTQ⁷¹ (SEQ ID NO: 22), ⁸⁰KVIVKLAD⁸⁷ (SEQ ID NO:23), ⁹⁷STVQMPIS¹⁰⁴ (SEQ ID NO: 24), ¹⁰⁶SWGGQVL¹¹² (SEQ ID NO: 25), and¹³⁴VSSSQEL¹⁴⁰ (SEQ ID NO: 26), of CfaB with the representative peptidesof CsfA (CS5), CstH (CS3), CsaB (CS4), CooA (CS1), CssA (CS6), and theCotA (CS2), we constructed a CFA multiepitope fusion antigen in silico.Web-based analyses suggested this putative CFA multiepitope fusionantigen possessed an antigenic propensity and hydrophilicity similar tothose of the native CFA/I major subunit CfaB. We then replacednucleotides coding these six peptides of the cfaB gene with nucleotidescoding the most antigenic peptide of each of these 6 CS subunit genes,and had a chimeric multiepitope CFA gene constructed.

The overlap of CFA/I/II/IV MEFA and the 3xSTa_(A14Q)-LT_(toxoid) or3xSTa_(N12S)-LT_(toxoid) PCR amplified products yielded twoCFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) chimeric genes (FIG. 1). Tworecombinant strains, 9208 and 9401, were constructed to express twoCFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) MEFA proteins. Two MEFA proteinsdiffered only at the STa toxoid, with STa_(A14Q) in 9208 and STa_(N12S)in 9401 (Table 1).

TABLE 1 E. coli strains and plasmid used in the present invention.Relevant properties Sources Strains BL21 B F⁻, ompT, hsdS GE Healthcare(r_(B) ⁻, m_(B) ⁻), gal, dcm. H10407 O78:H11; CFA/I, LT, STa JohnsHopkins Univ. EL 392-75 O6:H16; CS1/CS3, LT, STa Johns Hopkins Univ. UM75688 CS5/CS6, LT, STa Johns Hopkins Univ. E106 (E11881/9) CS4/CS6, LT,STa Univ. of Gothenburg E116 (E19446) CS3, LT, STa Univ. of GothenburgTHK38/pEU405 CS1 Emory Univ. DH5α/pEU588 CS2 Emory Univ. 9175pCFA/I/II/IV in BL21 Present invention 9164 p3xSTa_(A14Q)-tmLT in BL21Zhang et al., 2013 9318 p3xSTa_(N12S)-LT_(toxoid) in BL21 Presentinvention 9208 pCFA/I/II/IV- Present invention STa_(A14Q)-LT_(toxoid) inBL21 9401 pCFA/I/II/IV- Present invention STa_(N12S)-LT_(toxoid) in BL21Plasmids ρET28α Novagen pEU405 CS1 Emory Univ. pEU588 CS2 Emory Univ.pCFA/I/II/IV multiepitope CFA subunit Present invention gene in pET28αat NheI/EagI p3xSTa_(A14Q)- 3xSTa_(A14Q)-tmLT fusion Zhang et al., 2013tmLT gene in pET28α at NheI/EagI p3xSTa_(N12S)- 3xSTa_(N12S)-LT_(toxoid)fusion Present invention LT_(toxoid) gene in pET28α at NheI/EagIpCFA/I/II/IV- multiepitope CFA- Present invention STa_(A14Q)-LT_(toxoid) 2xSTa_(A14Q)-LT_(toxoid) in pET28α at NheI/EagIpCFA/I/II/IV- multiepitope CFA- Present invention STa_(N12S)-LT_(toxoid) 2xSTa_(N14s)-LT_(toxoid) in pET28α at NheI/EagI

Example 2

The CFA multiepitope fusion antigen was expressed as the 6×His-taggedprotein. DNA sequencing results showed each chimeric gene was a singleopen reading frame for a single 6×His-tagged CFA-_(toxoid) protein. Eachfusion protein consisted of 20amino acids from the pET28α vectorincluding the 6×His tag (six histidines), the CFA MEFA (150 amino acids)carrying epitopes of CFA/I and CS1-CS6 major subunits (CfaB, CooA, CotA,CstH, CsaB, CsfA and CssA), two copies of STa_(toxoid) STa_(A14Q) orSTa_(N12S), 109 amino acids (132-240) of dmLTA C-terminal peptide andone copy of the LTB subunit (100 amino acids), and four intra-peptidelinkers (FIG. 1C). The first copy of the STa_(toxoid) (without the stopcodon) with a ‘GPVD’ linker was inserted after the mutated 192th aminoacid residue (Arg-Gly) of the LTA, and the second STa_(toxoid) (with thestop codon) was at the C-terminus of the fusion protein with anL-linker.

SDS-PAGE with Coomassie blue staining showed over 90% of the6×His-tagged protein extracted from strains 9208 and 9401 had amolecular mass of about 48 KDa, the expected size of the 6×His-taggedCFAI/II/IV-STa-_(toxoid)-LT_(toxoid) MEFA protein. The 6×His-taggedprotein was recognized by anti-CFA/I and anti-CS1, -CS2, -CS3, -CS4,-CS5, and anti-CS6 MAb hybridoma supernatant, and rabbit anti-STa andanti-CT sera in Western blot assays (FIG. 1D).

Example 3

CFA/II/IV-STa-_(toxoid)-LT_(toxoid) MEFA proteins were well toleratedand immunogenic. T-84 cells incubated with 100 μg refolded fusionprotein CFA/I/II/IV-STa_(A14Q)-LT_(toxoid) orCFA/I/II/IV-STa_(N12S)-LT_(toxoid) showed no increase of intracellularcAMP and cGMP levels. That indicated that neither MEFA protein possesseddetectable LT or STa enterotoxicity. Additionally, female adult mice didnot display any noticeable adverse effects after i.p. immunization witheither CFA/I/II/IV-STa-_(toxoid)-LT_(toxoid) MEFA protein. Miceco-administrated with the multiepitope CFA/I/II/IV subunit protein andthe 3xSTa_(N12S)-LT_(toxoid) fusion also did not show any adverseeffects.

Female C57BL/6 mice did not display any apparent adverse effects afteri.p. immunized with this 6×His-tagged CFA multiepitope fusion antigenprotein. Mice immunized with CFA/I/II/IV-STa_(A14Q)-LT_(toxoid) orCFA/I/II/IV-STa_(N12S)-LT_(toxoid) developed immune responses to CFA/I,CS1, CS2, CS3, CS4/6, CS5/6, STa, and LT. Serum samples of the miceimmunized with fusion CFA/I/II/IV-STa_(A14Q)-LT_(taxi) had anti-CFA/I,-CS1, -CS2, -CS3, -CS4/6, -CS5/6, —STa, and anti-LT IgG antibodiesdetected at titers (in log₁₀) of 2.98±0.02, 2.92±0.06, 2.81±0.07,2.73±0.07, 2.84±0.03, 2.92±0.04, 2.40±0.75, and 3.19±0.01, respectively(FIG. 2). Serum samples from individual mice immunized with fusionCFA/I/II/IV-STa_(N12S)-LT_(toxoid) also developed IgG antibody responsesto CFA/I, CS1, CS2, CS3, CS4/6 and CS5/6 adhesins and both toxins (Table2). Serum of the mice i.p. immunized with the heat-extracted CFA/Ifimbriae had anti-CFA/I IgG detected at 2.95±0.01 (log₁₀).

There were no IgG antibodies specific to CFA/I, CFA/II, CFA/IV, STa orLT detected in the serum samples of the control mice or serum samplescollected prior to the primaryimmunization.

TABLE 2 Anti-CFA/I, -CS1, -CS2, -CS3, -CS4/CS5, and anti-CS5/CS6, andanti-STa and anti-LT IgG antibody titers (in log₁₀; mean ± standarddeviation) detected in the serum of mice immunized with theCFA/I/II/IV-STa_(N12S)-LT_(toxoid) or co-immunized with the CFA/I/II/IVMEFA and the 3xSTa_(N12S)-LT_(toxoid) toxoid fusion. Mouse immunizationMean serum IgG titer (log₁₀) ± stdev groups^(a) anti-CFA/I anti-CS1anti-CS2 anti-CS3 anti-CS4/6 anti-CS5/6 anti-STa anti-LT CFA/I/II/IV-2.63 ± 0.09 2.54 ± 0.04 2.44 ± 0.10 2.50 ± 0.09 2.58 ± 0.06 2.66 ± 0.032.50 ± 0.52 2.12 ± 0.30 STa_(N12S)- dmLT (n = 15) CFA/I/II/IV 2.69 ±0.05 2.56 ± 0.05 2.53 ± 0.08 2.50 ± 0.06 2.59 ± 0.03 2.67 ± 0.05 2.88 ±0.08 2.27 ± 0.13 plus 3xSTa_(N12S)- dmLT (n = 16) p 0.04 0.33 0.01 0.970.58 0.58 0.01 0.10 values^(b) ^(a)Groups of mice immunized with‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’, or co-immunized with ‘CFA/I/II/IV’and ‘3xSTa_(N12S)-LT_(toxoid)’. Anti-CFA/I, anti-CS1, anti-CS2,anti-CS3, anti-CS4/CS5 and anti-CS5/CS6, and anti-STa and anti-LT IgG inserum sample of each immunized mouse was titrated by ELISAs usingheat-extracted adhesin (500 ng adhesin per well of 2HB plates), STaconjugates (10 ng STa-ovalbumin conjugates per well of CoStar plates)and LT (100 ng LT per well of 2HB plates) as the coating antigen (intriplicates) and HRP-conjugated goat anti-mouse IgG (1:3300; Sigma) asthe secondary antibodies. IgG titers (in log₁₀) were expressed in means± standard deviation. ^(b)p values were calculated by using a Student ttest comparing mouse antibody titers in each group (15 mice immunizedwith CFA/I/II/IV-STa_(N12S)-LT_(toxoid), 16 mice co-immunized withCFA/I/II/IV and 3xSTa_(N12S)-LT_(toxoid)).

Mice immunized with CFA/I/II/IV-STa_(N12S)-LT_(toxoid) and miceco-immunized with CFA/I/II/IV and 3xSTa_(N12S)-LT_(toxoid) fusionsdeveloped similar or comparable levels of anti-adhesin and antitoxinantibody responses. Serum samples of the mice immunized with fusionCFA/I/II/IV-STa_(N12S)-LT_(toxoid) and the mice co-immunized withCFA/I/II/IV and 3xSTa_(N12S)-LT_(toxoid) developed similar levels of IgGantibody responses to CS1, CS3, CS4/6, CS5/6 and LT. Serum of theco-immunized mice (with CFA/I/II/IV and 3xSTa_(N12S)-LT_(toxoid)antigens) had greater titers of anti-CFA/I (2.69±0.05 vs 2.630.09),anti-CS2 (2.530.08 vs 2.440.10) and anti-STa (2.88±0.08 vs 2.50±0.52)IgG antibodies compared to the serum of the mice immunized withCFA/I/II/IV-STa_(N12S)-LT_(toxoid) (Table 2).

Example 4

Serum samples of the immunized mice were shown to inhibit adherence ofETEC strains expressing CFA/I, CS3, CS4/CS6, CS5/CS6 or CS6 and E. colistrains expressing CS1 or CS2 to Caco-2 cells. Serum samples pooled fromthe mice co-immunized with CFA/I/II/IV and 3xSTa_(N12S)-LT_(toxoid)exhibited significant inhibition activities against adherence of H10407(CFA/I⁺LT⁺STa⁺), E116 (CS3⁺LT⁺STa⁺), E106 (CS4⁺CS6⁺LT⁺STa⁺), UM75688(CS5⁺CS6⁺LT⁺STa⁺) and 2423/ETP98066 (CS6⁺LT⁺STa⁺), and E. colirecombinant strains expressing CS1 adhesin or CS2 adhesins (Table 3).Serum sample pooled from the mice immunized withCFA/III/IV-STa_(N12S)-LT_(toxoid) showed significant adherenceinhibition activities against all examined ETEC and E. coli strainsexcept the recombinant E. coli strain expressing CS2.

TABLE 3 Results of in vitro antibody adherence inhibition assays^(a),using serum samples of mice immunized withCFA/I/II/IV-STa_(N12S)-LT_(toxoid), co-administrated with CFA/I/II/IVMEFA and toxoid fusion 3xSTa_(N12S) -LT_(toxoid), or the negativecontrol mice. The number of ETEC or E. coli bacteria adhered to Caco-2cells incubated with pooled mouse serum and bacteria was used toindicate activity of anti- adhesin antibodies against bacteriaadherence. Mouse serum^(b) CFA/I/II/IV − CFA/I/II/IV + STa_(N12S)-3xSTa_(N12S)- Bacteria (CFUs) LT_(toxoid) LT_(toxoid) control H10407;CFA/I, 14.4 ± 14.8 39.7 ± 9.6  107.5 ± 9.2  LT, STa (×10⁴) p^(c) =0.0018 p = 0.017 THK38/pEU405; 18.3 ± 7.7  20.7 ± 11.6  77 ± 7.1 CS1(×10³) p = 0.012 p = 0.003 DH5a/pEU588; 13.5 ± 4.8  7.5 ± 4.5 16.5 ± 2.1CS2 (×10³) p = 0.28 p = 0.017 E116; CS3,  129 ± 49.9  86 ± 49.6  235 ±7.1 LT, STa (×10³) p = 0.003 p < 0.001 E106; CS4/CS6, 15.6 ± 3.3  26.7 ±3.9  125 ± 0  LT, STa (×10⁴) p < 0.001 p < 0.001 UM 75688; CS5/CS6, 59.5± 27.3  55 ± 35.2  110 ± 14.1 LT, STa (×10³) p = 0.029 p = 0.025ETP98066; CS6, 45.3 ± 28.6 53.3 ± 27.1 201.2 ± 29.5 LT, STa (×10⁴) p <0.001 p < 0.001 ^(a)ETEC field isolates and E. coli recombinant strainsexpressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 and CS6 (3.5 × 10⁶CFUs) were individually incubated with serum samples (20 μl) pooled frommice of the group immunized with CFA/I/II/IV-STa_(N12S)-LT_(toxoid)',the group co-immunized with the multiepitope CFA/I/II/IV and toxoidfusion 3xSTa_(N12S)-LT_(toxoid), or serum samples of the control mice ona shaker (50 rpm) for 1 hour at room temperature. The serum-bacteriamixture was added to Caco-2 cells (7 × 10⁵ cells; 1 ml final volume) andincubated in a CO₂ incubator for 1 hour. After washing off non-adherentbacteria, ETEC or E. coli bacteria adhered to Caco-2 cells (in 1 ml PBS)were serial diluted, plated, cultured overnight, and counted (CFUs).^(b)serum samples pooled from mice of the group immunized theCFA/I/II/IV-STa_(N12S)-dmLT, the group co-immunized with CFA/I/II/IV and3xSTa_(N12S)-LT_(toxoid), or the control group. These serum samples wereused in the antibody adherence inhibition assay. ^(c)p values werecalculated by using a Student t test comparing numbers of ETEC or E.coli bacteria adhered to the Caco-2 cells incubated with mouse serum ofeach immunization group vs. bacteria adherent to the cells treated withserum of the control group.

Example 5

Constructed CFA-toxoid multiepitope fusion antigen proteins carriedepitopes of 7 CFA adhesins, two copies of a STa toxoid, and a monomericLT_(A2-B) peptide (one copy of A2 peptide and one copy of B subunit toform a single peptide). Two recombinant strains, 9208 and 9401, wereconstructed to express two CFA-toxoid multiepitope fusion antigenproteins which differed only of the STa toxoid (Table 1). Both chimericgenes formed a single open reading frame, and expressed 6×His-taggedfusion proteins consisted of a CFA multiepitope fusion antigen (170amino acids) carrying epitopes of CFA/I and CS1-CS6 major subunits(CfaB, CooA, CotA, CstH, CsaB, CsfA and CssA), 2 copies of STa_(toxoid)STa_(A14Q) or STa_(N12S), 110 amino acids (131-240) of LT_(A1) peptide,and one copy of the LTB subunit (100 amino acids), and 4 intrapeptidelinkers (FIG. 5). The first copy of the STa toxoid, with a ‘GPVD’ linkerat the N-terminus, was inserted after the mutated LT_(A) 192th aminoacid residue (Arg→Gly), and the other copy of the STa_(toxoid) was atthe end of the fusion protein C-terminus. SDS-PAGE with Coomassie bluestaining showed over 90% of the His-tagged proteins extracted fromstrains 9208 and 9401 had a molecular weight of about 48 Kda, anexpected size of the 6×His-tagged CFAI/II/IV-STa_(toxoid)-LT_(toxoid)fusion protein. Refolded 6×His-tagged fusion proteins were recognized byrabbit anti-STa and anti-CT serum, and anti-CFA/I MAb.

Example 6

The CFA-toxoid multiepitope fusion antigen proteins were safe andimmunogenic. T-84 cells incubated with fusion proteinCFA/I/II/IV-STa_(A14Q)-LT_(toxoid) or CFA/I/II/IV-STa_(N12S)-LT_(toxoid)showed no increase of intracellular cAMP or cGMP levels. That indicatedthat both fusion proteins did not possess LT or STa enterotoxicity.Additionally, female adult mice did not display noticeable adverseeffects after i.p. immunization of either fusion protein, orco-administration of the CFA multiepitope fusion antigen protein with3xSTa_(N12S)-LT_(toxoid) toxoid fusion.

Mice immunized with CFA/I/II/IV-STa_(A14Q)-LT_(toxoid),CFA/I/II/IV-STa_(N12S)-LT_(toxoid), or co-administered with CFAmultiepitope fusion antigen and the 3xSTa_(N12S)-LT_(toxoid) toxoidfusion developed immune responses to CFA/I, CS1, CS2, CS3, CS4/6, CS5/6,STa, and LT. Serum samples from mice immunized with fusionCFA/I/II/IV-STa_(A14Q)-LT_(toxoid) had anti-CFA/I, -CS1, -CS2, -CS3,-CS4/6, -CS5/6, —STa, and anti-LT IgG antibodies detected at titers (inlog₁₀) of 2.98±0.02, 2.92±0.06, 2.81±0.07, 2.73±0.07, 2.84±0.03,2.92±0.04, 2.40±0.75, and 3.19±0.01, respectively (FIG. 6). Serumsamples from mice immunized with fusionCFA/I/II/IV-STa_(N12S)-LT_(toxoid) had IgG antibodies specific to CFA/I,CS1, CS2, CS3, CS4/6, CS5/6, STa, and LT detected at titers (in log₁₀)of 2.63±0.09, 2.54±0.04, 2.44±0.10, 2.50±0.09, 2.58±0.06, 2.66±0.03,2.5±0.52, and 2.12±0.30, respectively (FIG. 7). For the group of miceco-administered with the CFA multiepitope fusion antigen and the3xSTa_(N12S)-LT_(toxoid) toxoid fusion, anti-CFA/I, -CS1, -CS2, -CS3,-CS4/6, -CS5/6, —STa, and anti-LT IgG antibodies were titrated at2.69±0.05, 2.56±0.05, 2.53±0.08, 2.50±0.06, 2.59±0.03, 2.67±0.05,2.88±0.08, and 2.27±0.13(in log₁₀) from the serum samples (FIG. 8). Noanti-CFA, anti-STa, or anti-LT IgG antibodies were detected in serumsamples of the control mice, or serum samples collected prior toimmunization except one sample that was collected from one pre-immunizedmouse and had a low anti-CS4/CS6 IgG titer detected.

Example 7

Antibodies in serum samples of the immunized mice showed neutralizingactivities against STa toxin and CT in vitro. Pooled serum samples frommice immunized with ‘CFA/I/II/IV-STa_(A14Q)-LT_(toxoid)’,‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’, or co-immunized with ‘CFA/I/II/IV’and ‘3xSTa_(N12S)-LT_(toxoid)’ showed neutralization activity againstSTa (FIG. 9) and CT toxins (data not shown). Intracellular cGMP levelsin T-84 cells incubated with 2 ng STa toxin and serum of mice immunizedwith ‘CFA/I/II/IV-STa_(A14Q)-LT-_(toxoid)’,‘CFA/I/II/IV-STa_(N12S)-LT_(toxoid)’, and ‘CFA/I/II/IV’ with‘3xSTa_(N12S)-LT_(toxoid)’ were 36.7±20.7, 23.2±7.0, 0.31±0.36(pmole/ml), respectively. Whereas the cGMP levels in T-84 cellsincubated with 2 ng STa toxin and serum of the control mice or serumcollected pre-immunized mice were 80.7±7.3 and 75.1±11.2 (pmole/ml),which was equivalent to the cGMP in T-84 cells incubated with 2 ng STatoxin alone (78.7±7.2 pmole/ml; p=0.78, 0.63).

The cAMP levels in T-84 cells incubated with CT (10 ng) and the pooledserum sample of mice immunized with ‘CFA/I/II/IV-STa_(A14Q)-dmLT’,‘CFA/I/II/IV-STa_(N12S)-dmLT’, or ‘CFA/I/II/IV’ co-immunized with‘3xSTa_(N12S)-dmLT’ were 14.4±0.51, 12.8±0.76, and 7.7±2.4 pmole/ml,respectively (FIG. 16). These cAMP levels were significantly lower thanthose in cells incubated with the toxin alone (53.7±1.3; p<0.01)) orwith the toxin and the serum sample of the control mice (40.1±6.5;p<0.01).

Example 8

In accordance with some other embodiments, the present inventionprovides a modification of the CFA-STa_(N12S)-LT_(toxoid) MEFA of thepresent invention. This embodiment comprises 3 copies of the toxoidSTa_(N12S) in order to further enhance the fusion antigen anti-STaimmunogenicity, and it does not carry the histidine-tag so that the newantigen is suitable for human vaccine development. The nucleic acid andamino acid sequences are shown in FIGS. 17A-17C.

As shown in FIG. 18A, This CFA-toxoid MEFA carries 3 copies ofSTa_(N12S) at the N terminus, the C-terminus, and after the 192 residueof the LT A subunit gene, the CFA MEFA, and 160 to 192 AAs and 193 to240 AAs of the LT A, and the LT B subunit (see followed sequences andgenetic structure). Four linkers were included: GPGP (SEQ ID NO: 37),GGPVD (SEQ ID NO: 38), GPGP (SEQ ID NO: 37), and DPRVPSS (SEQ ID NO:39), to connect the STa_(N12S) with the LT, as well as the LTA with theLTB.

A Coomassie blue staining gel showed purity of the extractedhis-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA protein to be used toimmunize mice and pregnant sow (FIG. 18B). Western blotting showeddetection of this his-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA proteinby anti-CFA mAbs hybridoma supernatant (provided by Dr. AM Svennerholm),and anti-STa and anti-CT rabbit serum. Lane M, the protein marker; lane1, extracted proteins of 9419; lane 2, total protein of host E. coliBL21 (FIG. 18C).

The his-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA induces greatertiters of anti-STa antibodies in intraperitoneally immunized mice (FIG.19), and that induced anti-STa antibodies in mouse serum can completelyneutralize STa toxin (FIG. 20).

In addition, the double mutant LT (LT_(R192G/L211A); gift from PATH) wasfound an effective adjuvant to enhance the his-tag-lessCFA-3xSTa_(N12S)-LT_(toxoid) MEFA in inducing antibody responses to all7 adhesins and both toxins. For the first time this dmLT is an effectiveadjuvant in mouse parenteral immunization when combined with thehis-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA of the present invention(FIG. 21A-21H).

Example 9

The inventors carried out a pig immunization and challenge study toassess protective efficacy of the anti-STa antibodies induced by thehis-tag-less CFA-3xSTa_(N12S)-LT_(toxoid) MEFA against STa ETECinfection. Pregnant sows 6-8 weeks before farrowing was intramuscularlyimmunized with 500 μg of the his-tag-less CFA-3xSTa_(N12S)-LT_(toxoid)MEFA protein with 5 μg dmLT (as adjuvant). The immunized sows received abooster at the same dose two weeks before farrowing. A sow withoutimmunization was used as the control. Born piglets were orallychallenged with 5×10⁹ or 2×10¹⁰ CFUs of a STa ETEC recombinant strain8823 (pSTa/987P) after 24 hours suckling to acquire maternal antibodies.Challenged piglets were monitored every 3-4 hours during 24 hourpost-inoculation, and clinical symptoms including diarrhea, vomiting,dehydration and lethargy were recorded. In addition, each piglet wasweighed before and 24 hours after the challenge.

Immunized sow developed IgG and IgA antibodies in serum and colostrum(Table A). Piglets born by IM immunized sow acquired maternal anti-STa(& also anti-LT and anti-CFA) antibodies. When challenged with a STaETEC strain (5×10⁹ or 2×10¹⁰ CFUs), these piglets were protected as theyshowed no diarrhea (only 2 of 12had yellow pasty feces) (Table B), andmaintained daily weight gain (Table C). In contrast, piglets born thecontrol sow developed severe diarrhea (3 out 7challenged with 5×10⁹, and5out 6challenged with 2×10¹⁰) and showed daily weight loss.

TABLE A Sow colostrum and serum anti-STa IgG and IgA antibody titers (inlog₁₀) Pre- immunization Pre-booster Colostrum IgG Colostrum IgA SerumIgG serum IgG serum IgG cont immun cont immuni cont immun cont immuncont immun 0 3.07 ± 0.05 0 1.15 ± 0.03 0 2.32 ± 0.05 0 0 0 0

TABLE B Results from piglet challenge studies to show maternalantibodies against STa+ ETEC infection. A pregnant sow 7 weeks beforefallowing was IM immunized with 500 μg CFA-3xSTa_(N12S)-LT_(toxoid) with5 μg dmLT as adjuvant, and a booster at the same dose 2 weeks beforefallowing. A pregnant sow received no immunization severed as thecontrol. Piglets born by the immunized sow and the control sow werechallenged with STa+ ETEC strain 8823 (pSTa/987P) at day two, andeuthanized 24 h post-challenge. Severe Mild Challenge diarrhea diarrheadoses (with Watery (yellow (CFUs) Treatments dehydration) diarrhea pastyfeces) Healthy 5 × 10⁹ immunized 0 0 1 5 (n = 6) control 2 2 0 3 (n = 7)2 × 10¹⁰ immunized 0 0 1 5 (n = 6) control 1 4 0 1 (n = 6)

TABLE C Weight gain or loss (%) of piglets born by the immunized sow orthe control sow after challenged with the STa ETEC strain. challengedwith 5 × 10⁹ CFUs challenged with 2 × 10¹⁰ CFUs overall gain or losspiglets #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 mean stdev p valuecontrol 10 0 5.3 13 0 −6.7 5.5 −9.1 −23.1 0 −16.7 6.7 12.5 −0.2 11.10.01 (n = 13) immunized 9.7 9.4 11.8 7.7 6.3 3.8 9.5 14.3 6.3 7.1 10.5 88.7 2.8 (n = 12)

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A nucleic acid molecule encoding a fusion polypeptide moleculecomprising the colonization factor antigens (CFA) antigens CFA/I, CFA/II(CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6).
 2. The nucleic acid molecule ofclaim 1, wherein the nucleic acid molecule encoding the polypeptidecomprises the amino acid sequence of SEQ ID NO: 1, or having at least90% identity to SEQ ID NO:
 1. 3. The nucleic acid molecule of claim 1,further comprising a nucleic acid molecule encoding one or moreenterotoxins or fragments thereof covalently linked to the polypeptidemolecule.
 4. The nucleic acid molecule of claim 3, wherein theenterotoxins encoded by the nucleic acid molecule are selected from thegroup consisting of heat-labile toxin (LT), heat-stable toxin (STa). 5.The nucleic acid molecule of claim 4, wherein the nucleic acid moleculeencoding the polypeptide comprises nucleic acid molecules encoding atleast two enterotoxins or fragments thereof covalently linked to thepolypeptide molecule.
 6. The nucleic acid molecule of claim 4, whereinthe nucleic acid molecule encodes the amino acid sequence of SEQ ID NO:2 or having at least 90% identity to SEQ ID NO:
 2. 7. The nucleic acidmolecule of claim 4, wherein the nucleic acid molecule encodes the aminoacid sequence of SEQ ID NO: 3 or having at least 90% identity to SEQ IDNO:
 3. 8. The nucleic acid molecule of claim 5, wherein the nucleic acidmolecule encoding the polypeptide comprises three copies of heat-stabletoxin (STa) and one copy of heat-labile toxin (LT).
 9. The nucleic acidmolecule of claim 8, wherein the nucleic acid molecule encoding thepolypeptide comprises one or more copies of heat-stable toxin (STa)having either a A14Q mutation or a N12S mutation.
 10. The nucleic acidmolecule of claim 9, wherein the nucleic acid molecule encoding theheat-stable toxin (STa) has the N12S mutation.
 11. The nucleic acidmolecule of claim 10, wherein the nucleic acid molecule encodes an aminoacid sequence comprising the amino acid sequence of SEQ ID NO: 42 orhaving at least 90% identity to SEQ ID NO:
 42. 12. A nucleic acidmolecule having a nucleotide sequence selected from the group consistingof: SEQ ID NOS: 27-29, 40 and
 41. 13. An expression vector comprisingthe nucleic acid molecule of claim
 12. 14. A micro-organism transformedwith the expression vector of claim
 13. 15. A vaccine compositioncomprising the nucleic acid molecule of claim 12, and a pharmaceuticallyacceptable carrier.
 16. A method for treatment of EnterotoxigenicEscherichia coli (ETEC), infection in a subject comprising administeringto the subject an effective amount of a nucleic acid molecule encoding apolypeptide comprising one or more of the colonization factor antigens(CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)and/or comprising the amino acid sequence of SEQ ID NO: 1, or having atleast 90% identity to SEQ ID NO:
 1. 17. A method for treatment ofEnterotoxigenic Escherichia coli (ETEC), infection in a subjectcomprising administering to the subject an effective amount of a nucleicacid molecule encoding a polypeptide comprising the colonization factorantigens (CFA) antigens CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5,CS6) and further comprising heat-labile toxin (LT) and heat-stable toxin(STa) and/or comprising the amino acid sequence of SEQ ID NO: 2 and/or3.
 18. A method for treatment of Enterotoxigenic Escherichia coli(ETEC), infection in a subject comprising administering to the subjectan effective amount of a nucleic acid molecule encoding a polypeptidefusion polypeptide molecule comprising CFA antigens I, II and IV, STatoxoid and LTtoxoid.
 19. The method of claim 16, wherein the nucleicacid molecule encoding the fusion polypeptide is administered to thesubject in a vaccine formulation.
 20. The method of claim 16, furthercomprising administration of at least one additional therapeutic agent.21. The method of claim 20, wherein the additional therapeutic agent isselected from the group consisting of: anti-motility, antidiarrheals,antibiotics, antihelminths, and anti-parasiticals.
 22. The method ofclaim 17, wherein the nucleic acid molecule encoding the fusionpolypeptide is administered to the subject in a vaccine formulation. 23.The method of claim 17, further comprising administration of at leastone additional therapeutic agent.
 24. The method of claim 23, whereinthe additional therapeutic agent is selected from the group consistingof: anti-motility, antidiarrheals, antibiotics, antihelminths, andanti-parasiticals.
 25. The method of claim 18, wherein the nucleic acidmolecule encoding the fusion polypeptide is administered to the subjectin a vaccine formulation.
 26. The method of claim 18, further comprisingadministration of at least one additional therapeutic agent.
 27. Themethod of claim 26, wherein the additional therapeutic agent is selectedfrom the group consisting of: anti-motility, antidiarrheals,antibiotics, antihelminths, and anti-parasiticals.